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The InnoZyme™ TACE Activity Kit is a specific and sensitive assay designed to measure human TACE activity in cell lysates and biological samples and for screening enzyme inhibitors. An Anti-Human TACE-Coated 96-Well Plate is pre-coated with a monoclonal antibody specific for human TACE that captures the enzyme. Unbound material is discarded, the plate is washed, and the activity of captured TACE is measured using an internally quenched fluorescent substrate, MCA-KPLGL-Dpa-AR-NH2. Cleavage of the scissile amide bond, G-L, releases the fluorophore from the quenching molecule, Dpa, resulting in an increase in fluorescence. Fluorescence of the cleaved product, MCA-KPLG, is measured at an excitation wavelength of ~324 nm and emission wavelength of ~400 nm. The level of fluorescence is directly related to the enzyme activity.
• Highly specific: based on immunocapture of human TACE
• Highly sensitive and quantitative: assay range 5-100 ng/ml
• Convenient: 96-well format and non-radioactive detection
• Versatile: appropriate for measuring active TACE and high-throughput screening of TACE inhibitors
The activity of increasing concentrations human recombinant TACE (Cat. No. PF133) was measured according to the Detailed Protocol above. Human colorectal adenocarcinoma cells, DLD1 and HT-29 and human glioblastoma cells, T98G, were cultured in DMEM medium supplemented with 10% FCS and harvested at 80-90% confluency. Total cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) and the manufacturer's recommended protocol. TACE activity was measured according to the Detailed Protocol above. RFU is reported per mg protein.
Materials Required but Not Delivered
• Distilled H2O • Pipettors or multi-channel pipettor precisely calibrated to the target volume • 37°C incubator • Fluorescent plate reader capable of measuring fluorescence at excitation 320-330 nm and emission 400-410 nm • TACE, His·Tag®, Human, Recombinant, T. ni (Cat. No. PF133) (optional; for inhibitor screening)
References
References
Kirkegaard, T., et al. 2004. Clin. Exp. Immunol.135, 146. Neumann, U., et al. 2004. Anal. Biochem.328, 166. Skovronsky, D.M., et al. 2001. J. Neurobiol.49, 40. Black, R.A., et al. 1997. Nature385, 729. Moss, M.L., et al. 1997. Nature385, 733.
5-100 ng/ml as measured with purified recombinant human TACE
Sample Type
Cells
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Intended use
The InnoZyme™ TACE Activity Kit is designed to measure human TACE activity in cell lysates and biological samples. The assay may also be used for screening enzyme inhibitors, but the end-user must supply purified enzyme for this application.
Storage and Shipping Information
Ship Code
Dry Ice Only
Toxicity
Multiple Toxicity Values, refer to MSDS
Storage
Multiple storage requirements
Storage Conditions
Upon receipt the unopened kit should be stored at -20°C or -70°C. All kit components, once opened, can be stored for up to 3 months under the following conditions:
Upon receipt the unopened kit should be stored at -20°C or -70°C. All kit components, once opened, can be stored for up to 3 months under the following conditions:
*Note: Following initial use the Control should be dispensed into aliquots and stored at -70°C. Avoid freeze/thaw cycles.
Intended use
The InnoZyme™ TACE Activity Kit is designed to measure human TACE activity in cell lysates and biological samples. The assay may also be used for screening enzyme inhibitors, but the end-user must supply purified enzyme for this application.
Background
TACE (TNF-αConverting Enzyme), also known as ADAM17 or α-secretase, is a membrane-anchored zinc protease expressed in its active form on the surface of several cell types. The enzyme belongs to the ADAM family of proteins, which are multi-domain proteins consisting of a signal sequence, a prodomain, metalloprotease-like, disintegrin-like, cysteine-rich, and EGF-like repeat domains, followed by a transmembrane region and cytoplasmic tail. The most well known physiological substrate for TACE is pro-TNF-α, the membrane-bound precursor of TNF-α, which is a potent pro-inflammatory cytokine. Active TNF-α is released from the membrane and mediates the recruitment and activation of inflammatory cells to injured or infected tissues. Elevated levels of circulating TNF-α have been demonstrated in several acute and chronic pathological conditions, such as LPS-induced septic shock, arthritis, pleurisy, Crohn's disease, and inflammatory bowel disease. TACE is also responsible for the proteolytic cleavage of amyloid precursor protein (APP), the protein from which amyloid β-peptides are derived. β-peptides are among the major components of senile plaques, the aberrant structures that are present in the brain of Alzheimer's disease (AD) patients. TACE was found to localize with senile plaques and neurofibrillary tangles in the hippocampus and cortex of brains from AD patients. In addition to pro-TNF-α and APP, TACE is also known to cleave p55, p75 TGF-α, interleukin-1R-II, and L-selectin.
Principles of the assay
The InnoZyme™ TACE Activity Kit is a specific and sensitive assay for measuring active human TACE. The Anti-Human TACE-Coated 96-Well Plate is pre-coated with a monoclonal antibody specific for human TACE that captures the enzyme. Unbound material is discarded, the plate is washed, and the activity of captured TACE is measured using an internally quenched fluorescent substrate, MCA-KPLGL-Dpa-AR-NH2. Cleavage of the scissile amide bond, G-L, releases the fluorophore from the quenching molecule, Dpa, resulting in an increase in fluorescence. Fluorescence of the cleaved product, MCA-KPLG, is measured at an excitation wavelength of ~324 nm and emission wavelength of ~400 nm. The level of fluorescence is directly related to the enzyme activity.
Materials provided
Important: please note that the quanity of control supplied with the assay is sufficient only for use as a control sample and will not cover the entire plate.
• Anti-Human TACE-Coated 96-Well Plate (Kit Component No. JA9118): 96-well polystyrene plate supplied as 6 strips, 2 X 8 wells each, coated with an antibody specific for human TACE, 1 each • Control (Kit Component No. JA9119): human recombinant TACE, 1 vial • Substrate (Kit Component No. JA9120): MCA-KPLGL-Dpa-AR-NH2, 60 µl, 2 mM in DMSO, 1 vial • Sample Buffer (Kit Component JA9122): 20 ml, 1 bottle • Assay Buffer (Kit Component JA9121): 20 ml, 1 bottle • Wash Buffer (Kit Component No. JA9123): 20 ml, supplied as 20X, 1 bottle • Plate Sealer: 1 each
Materials Required but not provided
• Distilled H2O • Pipettors or multi-channel pipettor precisely calibrated to the target volume • 37°C incubator • Fluorescent plate reader capable of measuring fluorescence at excitation 320-330 nm and emission 400-410 nm • TACE, His·Tag®, Human, Recombinant, T. ni (Cat. No. PF133) (optional; for inhibitor screening)
Preparation
Dilute samples as necessary with Sample Buffer.
Recommended guidelines for sample preparation:
1. Total protein concentration in cell lysates should be >5 mg/ml
2. Dilution factor for cell lysates at >5 mg/ml is approximately 1:20 or higher
Reagent preparation
All reagents necessary to perform the assay are supplied with the kit. Warm all reagents to room temperature (15-25°C) immediately prior to use.
For analyzing biological samples the following reagent preparation is required:
• Wash Buffer (1X): Dilute Wash Buffer 1:20 with dH2O . To prepare Wash Buffer (1X) for a single wash step for the entire plate, add 2 ml Wash Buffer to 38 ml dH2O.
• Control: Dilute the Control with chilled Sample Buffer. Refer to the vial label for lot-specific dilution information.
Important Note: Following initial thaw the Control should be dispensed into aliquots and stored at -70°C. Avoid freeze/thaw cycles.
• Substrate (1X): Dilute Substrate 1:200 with Assay Buffer. To prepare Substrate (1X) for the entire plate, add 50 µl Substrate to 9.95 ml Assay Buffer.
For inhibitor screening the following additional reagent preparation is required:• Test inhibitor(s): Prepare dilutions of test inhibitors in Assay Buffer as necessary.
• Diluted Substrate: Dilute Substrate 1:50 with Assay Buffer. To prepare Substrate for the entire plate add 40 µl Substrate to 1.95 ml Assay Buffer.
Detailed protocol
Note: It is recommended that all samples and controls be assayed in duplicate.
Recommended Protocol for Assessing Activity in Biological Samples
1. Remove the desired number of strips from the Anti-Human TACE-Coated 96-Well Plate. Return the remaining strips to the re-sealable foil pouch, seal the edge, and store at 4°C. 2. Wash the plate by adding 350-400 µl Wash Buffer (1X) to each well. Following the wash discard the contents of the wells by shaking the contents of the wells into the sink; invert the plate and tap on a paper towel to remove residual liquid. Repeat for a total of 2 washes. 3. Add 100 µl of each Control or sample to individual, designated wells; prepare a Blank by adding 100 µl Sample Buffer to designated wells. 4. Cover the plate with the Plate Sealer and incubate 1 h at room temperature with gentle shaking. 5. Wash the plate by adding 350-400 µl Wash Buffer (1X) to each well. Following the wash discard the contents of the wells by shaking the contents of the wells into the sink; invert the plate and tap on a paper towel to remove residual liquid. Repeat for a total of 5 washes. 6. Add 100 µl Substrate (1X) to each well and cover the plate with the Plate Sealer. Incubate 4-5 h at 37°C. Note: seal the plate tightly to prevent evaporation. Any changes in the reaction volume can influence the fluorescence reading. 7. Read and record the fluorescence measured at an excitation wavelength of ~324 nm and an emission wavelength of ~405 nm.
Protocol Modifications for Inhibitor Screening
1. Following the wash cycles in step 5 add 100 µl Test Inhibitor (specifically prepared for inhibitor screening) to designated wells and cover the plate with the Plate Sealer. Incubate the plate for the desired amount of time at room temperature with gentle shaking. 2. In place of 100 µl Substrate (1X), add 20 µl Diluted Substrate (specifically diluted for inhibitor screening) to each well and gently shake the plate to mix. Incubate 4-5 h at 37°C. Proceed to step 7.
Calculations
The results are displayed in relative fluorescence units (RFU):
1. Correct the fluorescence from each sample and Control by subtracting the fluorescence of the Blank.
2. Calculate the mean fluorescence for each sample and Control from duplicate readings to obtain the final RFU.
Assay characteristics and examples
Figure 1: Activity of Recombinant Human TACE
The activity of increasing concentrations human recombinant TACE (Cat. No. PF133) was measured according to the Detailed Protocol above.
Figure 2: Activity of TACE/ADAM17 in Biological Samples
Human colorectal adenocarcinoma cells, DLD1 and HT-29 and human glioblastoma cells, T98G, were cultured in DMEM medium supplemented with 10% FCS and harvested at 80-90% confluency. Total cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) and the manufacturer's recommended protocol. TACE activity was measured according to the Detailed Protocol above. RFU is reported per mg protein.
Assay Range
5-100 ng/ml as measured with purified recombinant human TACE
Registered Trademarks
Calbiochem® is a registered trademark of EMD Chemicals, Inc. CytoBuster™, Interactive Pathways™ and InnoZyme™ are trademarks of EMD Chemicals, Inc.
